Neovascular-targeted immunoconjugates

ABSTRACT

Immunoconjugates for treating diseases associated with neovascularization such as cancer, rheumatoid arthritis, the exudative form of macular degeneration, and atherosclerosis are described. The immunoconjugates typically consist of the Fc region of a human IgG1 immunoglobulin including the hinge, or other effector domain or domains that can elicit, when administered to a patient, a cytolytic immune response or cytotoxic effect against a targeted cell. The effector domain is conjugated to a targeting domain which comprises a factor VII mutant that binds with high affinity and specificity to tissue factor but does not initiate blood clotting such as factor VII having a substitution of alanine for lysine-341 or of alanine for serine-344.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of PCT/US00/16481, filed Jun. 14, 2000, which claims benefit of U.S.Provisional application No. 60/142,161, filed Jul. 1, 1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was made with partial government support under ProjectGrant HL29019-17 from the National Institutes of Health, U.S. PublicHealth Service. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the design, synthesis, and administration ofimmunoconjugate reagents for treating patients having diseasesassociated with the growth of new blood vessels (neovascularization)such as cancer, rheumatoid arthritis, the exudative form of maculardegeneration, and atherosclerosis. The target for immunoconjugates ofthe invention is the transmembrane receptor tissue factor expressed byendothelial cells of the neovasculature. Tissue factor also is expressedby many types of tumor cells. Therefore, therapeutic methods of theinvention are especially efficacious in immunotherapy against a broadrange of solid tumors. The therapeutic reagent is an immunoconjugatecomposed of a targeting domain and an effector domain (FIG. 1). Thetargeting domain is a mutated form of factor VII that binds with highaffinity and specificity to tissue factor, but does not initiate bloodcoagulation. The effector domain is the Fc region of an IgG1immunoglobulin.

2. Background of the Invention

Pathologic angiogenesis, the induction of the growth of blood vesselsfrom the vessels in surrounding tissue, is observed in a variety ofdiseases, typically triggered by the release of specific growth factorsfor vascular endothelial cells. Pathologic angiogenesis can result inneovascularization, enabling solid tumor growth and metastasis, causingvisual malfunction in ocular disorders, promoting leukocyteextravasation in inflammatory disorders, and/or influencing the outcomeof cardiovascular diseases such as atherosclerosis. Collectively, theseare sometimes referred to as angiogenic diseases.

Since the survival and growth of a solid tumor depend critically on thedevelopment of a neovasculature, cancer is a paramount angiogenicdisease (Folkman, J. (1995) N. Engl. J. Med. 333, 1757-1763). Manycancers progress in stages, beginning with proliferation of primarytumor cells, then penetration of tumor cells into the circulatorysystem, colonization at disseminated metastatic sites, and proliferationof the metastasized tumor cells which are responsible for most deathsfrom cancer (Vogelstein, B., and Kinzler, K. W. (1993) TIG 9, 141-143).Because cancer often remains undetected until the disease has reachedthe metastatic stage, cancer therapies that can eradicate the vascularinfrastructure and metastatic tumor cells are particularly desirable.

Angiogenesis also plays a significant role in rheumatoid arthritis(Szekanez, Z., et al. (1998) J. Invest. Med. 46, 27-41). Rheumatoidarthritis (RA) is a chronic systemic inflammatory disease that occursworldwide in all ethnic groups and predominantly affects diarthrodialjoints and frequently a variety of other organs. The RA synovial tissueis extensively neovascularized. In RA, inflammatory leukocytes emigrateinto the synovium through the endothelial layer of blood vessels,resulting in synovial inflammation and, eventually, joint destruction.

Angiogenesis underlies the majority of eye diseases that result incatastrophic loss of vision (Friedlander, M., et al. (1996) Proc. Natl.Acad. Sci. USA 93, 9764-9769). The leading cause of blindness inindividuals over the age of 55 is the exudative (“wet”) form ofage-related macular degeneration (ARMD), and under the age of 55,proliferative diabetic retinopathy (PDR). While ARMD and PDR areprototypic diseases for choroidal and retinal neovascularization,respectively, other degenerative or inflammatory conditions canselectively cause angiogenesis of either vasculature (ibid.).

Therefore, one approach to the treatment of these disease states, andparticularly of cancer, has been to compromise the function or growth ofthe neovasculature, primarily by inhibiting the growth of new bloodvessels (Chaplin, D. J., and Dougherty, G. J., (1999) Br. J. Cancer, 80,57-64). There are several advantages to vascular targeting. First,damage to blood vessels could stop blood flow and, applied to cancer,can trigger death of many dependent tumor cells. Second, the targetcells are adjacent to the bloodstream, enhancing drug delivery. Third,treatment-resistant mutations are not likely to emerge in vascularendothelial cells.

A number of anti-angiogenic therapies have been suggested, includingdrug, antibody, and gene therapy-based approaches. These includemetalloproteinase inhibitors, pentosan polysulphate and TNP-470,selective inhibitors of tyrosine kinase, and peptide inhibitors ofangiostatin and endostatin (ibid., and reviews cited therein). Thesetypically prevent angiogenesis at various stages of vessel formation,i.e., basement membrane degradation, endothelial cell migration,endothelial cell proliferation, and tube formation. It would bedesirable to have improved therapies that target not only angiogenesis,but also the neovasculature already formed in angiogenic disease states.

SUMMARY OF THE INVENTION

It is an objective of the invention to provide a novel treatment fordiseases involving the growth of new blood vessels (neovasculature),including cancer, rheumatoid arthritis, the exudation form of maculardegeneration, and athlerosclerosis. It is another and more specificobjective of the invention to provide a neovascular-targeted therapythat not only inhibits the angiogenesis observed in these diseasestates, but also destroys the neovasculature structure.

These and other objectives are accomplished by the present invention,which provides compositions comprising an immunoconjugate containing atargeting domain that binds selectively to tissue factor and an effectordomain that mobilizes a cytolytic immune response or cytotoxic responseagainst a targeted cell. In a typical immunotherapy treatment, acomposition containing at least one immunoconjugate constructed as adimer of two identical chains, each having an effector domain which isthe Fc region of a human IgG1 immunoglobulin, including the hinge,conjugated to a targeting domain comprising a mutant form a human factorVII such as a factor VII with a substitution of alanine for lysine-341and/or alanine for serine-344, that binds to tissue factor but does notinitiate blood clotting, is administered in effective amounts to apatient having a disease associated with neovascularization. Sincetissue factor is expressed by endothelial cells lining the tumorneovasculature but not the normal vasculature, and also by many types oftumor cells, factor VII immunoconjugates of the invention are especiallyefficacious in immunotherapy against a broad range of solid tumors. Theinvention is different from previously described inventions whichactivate coagulation in the neovasculature and/or introduce tissuefactor or tissue factor mutants to the neovasculature (such as thatdescribed by Edgington and Morrissey in U.S. Pat. No. 6,001,978), whichis superfluous because tissue factor is specifically expressed by theendothelial cells of the neovasculature, in two major respects: tissuefactor is used as a target for inducing a cytolytic immune responsemediated by immunoconjugates of the invention, rather than as aninitiator of the blood coagulation process, and a cytolytic immuneresponse that destroys neovasculature without activating the bloodcoagulation pathway is initiated.

Methods for systemic or local administration of the immunoconjugates arealso disclosed, which involve the use of purified immunoconjugateproteins in conventional pharmaceutical compositions, or vector systemscarrying a cDNA encoding a secreted form of the immunoconjugate.Treatments according to the invention include, but are not limited to,periodic or continuous intravenous or intratumoral injection, orinjection at other sites, of effective amounts of one or more types ofpurified immunoconjugate protein. Alternate embodiments involve thetreatment of patients by intravenous or intratumoral injection, orinjection at other sites, of an effective amount of an expression vectorcarrying a cDNA encoding a secreted form of one or more types ofimmunoconjugate protein. Examples of the latter include treatment ofpatients by intravenous or intratumoral injection, or injection at othersites, of a replication-deficient adenoviral vector or anadeno-associated vector carrying a cDNA encoding a secreted form ofimmunoconjugates of the invention.

In some embodiments such as cancer immunotherapy, immunoconjugates ofthe invention are administered together with another type ofimmunoconjugate having a different targeting domain. These typically aresingle-chain Fv or V_(H) molecule fragments isolated from a human scFvor V_(H) fusion phage libraries that bind selectively to cell surfacemolecules expressed on tumor cells, vascular endothelial cells, invasivecells in the synovium in pathological conditions, and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram illustrating the organization of an immunoconjugatemolecule of the invention. TD: Targeting domain. H: Hinge region of anIgG1 immunoglobulin with 2 disulfide bridges. CH2 and CH3: Constantregions of an IgG1 immunoglobulin. The targeting domain typicallyconsists of human factor VII with a mutated active site or a human scFvor V_(H) molecule. The effector domain typically consists of the Fcregion of a human IgG1, unmodified or conjugated to a cytotoxic agentsuch as a radioactive molecule or photoactivateable dye molecule.

FIG. 2 is a bar graph showing immunoconjugate-dependent lysis of humanmelanoma cells by human natural killer (NK) cells. The targeting domainof the immunoconjugate is a melanoma-specific human scFv molecule, andthe effector domain is the Fc region of a human IgG1 immunoglobulin. Thehuman melanoma cell line A-2058 and the human fibroblast cell controlwere labeled with the fluorescent dye Calcein-AM. The fraction ofmelanoma or fibroblast cells remaining intact after exposure to NK cellsalone (Bar A), or to NK cells with the scFv immunoconjugate E26-1 (BarB), was measured by residual fluorescence. The ratio of NK effectorcells to target cells (E/T) was varied from 3 to 20. Three complete setsof experiments were done for both the melanoma and fibroblast cells; foreach experiment, the cytolysis assays were done in quadruplicate. Thebars shown in the figure represent the average of the cytolysis assaysfor the three experiments, which generally agreed with 10%. The %cytolysis was calculated as described in Example 1. Similar results wereobtained with the scFv immunoconjugate G71-1.

FIG. 3 is a bar graph showing immunoconjugate-dependent lysis of humanmelanoma cells by complement. The procedure was as described in thelegend of FIG. 2, except that human serum or purified rabbit complementcomponents were used instead of NK cells. Immunoconjugate and complementreagents for each assay were as shown in Example 1. Table 1.

FIG. 4 shows a immunohistochemical assay for binding of a mutated mousefactor VII (mfVIIasm) immunoconjugate to tumor cells and tumor vascularendothelial cells in a human melanoma xenograft grown in SCID mice. The2nd antibody was anti-human γ-chain labeled with AP, and the APsubstrate was BCIP/NBT which produces a blue color; the counterstain wasmethyl green. Panel A: Control stained with hematoxylin+eosin showingextensive vascularization of the xenograft. Panel B:Immunohistochemistry with the mfVIIasm immunoconjugate showing intensestaining of both the vascular endothelial cells and tumor cells. PanelC: Immunohistochemical control without the mfVIIasm immunoconjugate.

FIG. 5 are line graphs showing concentrations of the G71-1 scFv andmfVIIasm immunoconjugates in the blood of SCID mice after intravenousinjections of replication-incompetent adenoviral vectors encoding theimmunoconjugates. The mice were injected on day 0 and day 7 with 2×10¹¹adenovirus encoding the G71-1 immunoconjugate (A) or with 4×10¹¹adenovirus encoding the mfVIIasm immunoconjugate (B). The concentrationof the encoded immunoconjugate in the blood was determined by ELISA.Each point is the average of the concentration for the 5 mice in eachgroup.

FIG. 6 is a line graph showing the inhibitory effect of the G71-1 andmfVIIasm immunoconjugates on the growth of a human melanoma xenograft inSCID mice. For each curve 5 SCID mice were injected subcutaneously with5×10⁵ TF2 cells. When the xenografts had grown to a palpable size, themice received tail vein injections on days 0, 7 and 14 of theadenoviruses indicated in the figure. The amount of adenovirus injectedwas 4×10¹¹ for the control, 2×10¹¹ for the adenovirus encoding the G71-1immunoconjugate and 4×10¹¹ for the adenovirus encoding the mfVIIasmimmunoconjugate. The estimated tumor volumes are the averages for the 5mice in each group.

FIG. 7 is a bar graph showing tumor weights of the xenografts from theexperiment reported in FIG. 6 and Example 2. The xenografts weredissected from the mice on day 20, which was 6 days after the lastinjection of adenovirus. The bar heights are the average weights for the5 mice in each group.

FIG. 8 is a line graph showing the inhibitory effect of the G71-1 andmfVIIasm immunoconjugates on the growth of a large human melanomaxenograft in SCID mice. Each mouse was injected subcutaneously with5×10⁵ melanoma cells, and the xenografts were allowed to grow to anestimated tumor volume of 50 mm³ on the skin surface (day 1). A mixtureof 2×10¹¹ adenoviruses encoding the G71-1 immunoconjugate and 7×10¹¹adenoviruses encoding the mfVIIasm immunoconjugate was injected into thetail vein of 5 mice on days 1, 6, 12 and 19. As a control 5 mice wereinjected with 4×10¹¹ adenoviruses that did not encode animmunoconjugate. The estimated tumor volumes are the averages for the 5mice in each group. One of the mice injected with the adenovirusesencoding the immunoconjugates was found dead on day 17; the estimatedtumor volumes on subsequent days are the averages for the remaining 4mice.

FIG. 9 is a line graph plotting the inhibitory effect of the mfVIIasmimmunoconjugate on the growth of a human melanoma xenograft expressing alow level of tissue factor. The mice was injected subcutaneously with5×10⁵ LXSN cells, and when the xenograft had grown to a palpable size(day 0) 5 mice were injected intravenously with 9×10¹¹ adenovirusesencoding the mfVIIasm immunoconjugate, and 5 mice were injected with4×10¹¹ control adenoviruses. Additional injections were done on days 7,13, 21 and 24, and on day 25 the mice were dissected for morphologicaland histochemical examination. The estimated tumor volumes are theaverages for the 5 mice in each group.

FIG. 10 illustrates the histochemistry of the human melanoma xenograftsfrom the experiment reported in the legend of FIG. 9 and Example 2. Thexenografts were dissected on day 25, embedded in paraffin, and sectionswere stained with hemotoxylin×eosin. Panel A: Xenograft from a controlmouse injected with the adenovirus that does not encode animmunoconjugate. Panel B: Xenograft from a mouse injected with theadenovirus encoding the mfVIIasm immunoconjugate.

FIG. 11 is a line graph showing dosage effect of intratumorally injectedadenoviral vectors on the growth of a human melanoma tumor in SCID mice.The mice were first injected subcutaneously with the human melanoma cellline TF2, and when a skin tumor had grown to the size indicated on day 0the tumor was injected with a mixture of the two adenoviral vectorsencoding the mfVIIasm and G71-1 immunoconjugates. The dose for the twovectors was as follows. ◯: 7×10⁸ IU; ∇: 2×10⁹ IU; □: 6×10⁹ IU. The dosefor the control, ⋄, was injected with 6×10⁹ IU of an adenoviral vectorthat did not encode an immunoconjugate.

FIG. 12 is a line graph showing the effect of a high dose ofintra-tumorally injected adenoviral vectors on the growth of a humanmelanoma tumor in SCID mice. The mice were first injected subcutaneouslywith the human melanoma line TF2, and when a skin tumor had grown to thesize indicated on day 0 the tumor was injected with 6×10⁹ IU of thefollowing adenoviral vectors: Control vector (3 mice); ▪, vectorencoding the mfVIIasm immunoconjugate (5 mice;

, mixture of the two vectors encoding the mfVIIasm and G71-1immunoconjugates (6 mice). Additional injections were done on days 2, 5,7, 9, 11, 13, 15 and 17. The points on each curve are the average (±20%)of the measurements for all mice in the corresponding group.

FIG. 13 are photographs of melanoma tumors from SCID mice injectedintratumorally with the adenoviral vectors encoding a mixture of themfVIIasm and G71-1 immunoconjugates or with an empty adenoviral vectorcontrol. The experiment is described in the legend to FIG. 12 above, andin Example 3. The tumors were dissected 2 days after the last injectionand photographed. The full scale at the top of the figure is 1 cm.

FIG. 14 are photographs showing the distribution of an adenoviral vectorin tumor and liver after intratumoral injection. The control vectorencoding the GFP protein but not an immunoconjugate was injected into 3sites of a human melanoma skin tumor growing in SCID mice. The totalvector dose was 6×10⁹ IU. The tumor and liver were dissected 40 hr afterthe injection and were examined intact under a dissecting microscopewith fluorscence optics. The GFP signal was detected with 480 nmexcitation and 630 nm emmision, and the background signal was detectedwith 577 nm excitation and 630 nm emission. Panel A: Tumor GFP. Panel B:Tumor background. Panel C: Liver GFP. Panel D: Liver background. Abright fluorescent spot similar to the one in Panel A also was detectedat two other tumor sites, presumably corresponding to the injectionsites. The photographs are focused at one level in the tissues. However,the GFP spot in the tumor also could be detected by focusing above andbelow that level, suggesting that the tumor cells adjacent to the pathtraversed by the injection needle are the only cells injected by thevector.

FIG. 15 shows liver sections from SCID mice injected intravenously orintratumorally with the adenoviral vectors. The intravenous experimentis described in Example 2, and the intratumoral experiment is describedin Example 3. The panels on the left were injected with the controlvector, and the panels on the right were injected with a mixture of thetwo vectors encoding the mfVIIasm and G71-1 immunoconjugates. The liverswere dissected 2 days after the last injection, fixed in formaldehydeand embedded in paraffin. The sections were stained with hematoxylin andeosin and photographed at a magnification of 100×.

FIG. 16 is a line graph showing the effect of intravenous injectionsinto immunocompetent mice of the adenoviral vector encoding the mfVIIasmimmunoconjugate on the growth of a mouse skin melanoma. C57BL/6 micewere injected subcutaneously with the mouse melanoma line B16F10, andwhen a skin tumor had grown to the size indicated on day 0 the tumor wasinjected with 3×10¹⁰ IP of the vector encoding the mfVIIasmimmunoconjugate. The Fc domain of the immunoconjugate was derived from amouse IgG1 immunoglobulin. The tumor was injected again on day 5 with1.5×10¹⁰ IP.

DETAILED DESCRIPTION OF THE INVENTION

Anti-vasculature immunoconjugate therapy is based on the observationthat normal adult mammalian vasculature is generally in a quiescentstate (except for certain processes such as the female reproductivecycle and wound healing), in contrast to the neovasculature that formsin certain disease states such as a growing tumor which is in an activestate of angiogenesis. Therefore, any molecular difference betweenquiescent and proliferating vascular endothelial cells could serve as atarget for the pathologic vasculature.

A switch from a quiescent state to an angiogenic state in the pathologicvasculature such as that observed in cancer is usually activated byvascular endothelial growth factor (VEGF), which is secreted by tumorcells and binds with high affinity and specificity to VEGF receptors onvascular endothelial cells. Another response activated by the binding ofVEGF to receptors on vascular endothelial cells is the expression oftissue factor, a transmembrane receptor that binds plasma factorVII/VIIa to initiate blood coagulation. Because only the vascularendothelial cells that have bound VEGF express tissue factor, a putativetarget for the tumor vasculature is tissue factor expressed onendothelial cells which should bind factor VII/VIIa circulating in theblood.

This invention is based upon the finding that immunoconjugates composedof a targeting domain conjugated to the Fc domain of human IgG1 mediatea cytolytic response against targeted cells by the natural killer (NK)cell and complement pathways of the immune system (Wang, B., et al.(1999) Proc. Natl. Acad. Sci. USA 96, 1627-1632). Since the bindingbetween the cell surface receptor tissue factor expressed the innersurface of growing blood vessels, but not of the stable blood vesselspresent in normal tissues, and its natural ligand factor VII exhibitshigh specificity and affinity (Hu, Z., et al. (1999) Proc. Natl. Acad.Sci. USA 96, 8161-8166), the invention provides a new immunotherapyprotocol for the treatment of diseases associated withneovascularization. The invention also provides enhanced efficacy forcancer treatments because tissue factor is also expressed by many typesof tumor cells (Callander, N. S., et al. (1992) Cancer, 70, 1194-1201).

However, because the binding of a factor VII immunoconjugate to tissuefactor might cause disseminated intravascular coagulation, factor VIImutants that inhibit coagulation without affecting affinity for tissuefactor are employed in preferred embodiments of the invention. Theseimmunoconjugates that bind to tissue factor but do not initiate bloodclotting compete with endogenous factor VII/VIIa for binding to tissuefactor. This competitition strongly favors the immunoconjugate (Hu, etal., cited above) because it contains two factor VII targeting domains(FIG. 1), providing an avidity effect lacking in the monomericendogenous factor VII molecule. In many preferred embodiments, theactive site of human factor VII is mutated by site-directed mutagenesis,substituting alanine for Lys-341 and/or for Ser-344, in order to blockthe proteolytic activity that initiates the blood coagulation processwhen factor VII binds to tissue factor. Because both the targeting andeffector domains can be derived from human sources, significant immunerejection responses in human patients are minimized.

In the generalized practice of the invention, immunoconjugates such asthat set out in FIG. 1 are constructed as a protein dimer comprising twochains, each having an effector domain conjugated to a targeting domain.Several type sof effector domain may be employed, so long as theseexhibit cytotoxicity upon binding of the immunoconjugate to its target.Many typical immunoconjugate proteins of the invention have, as aneffector domain, the Fc region of an IgG1 immunoglobulin. As usedherein, this includes variants and truncated versions exhibiting thesame biological function as the Fc region. In other embodiments, theeffector domain can be a cytotoxic agent such as a radioactive tag orphotoactivateable dye molecule, e.g., a dye that can be activated by alaser beam. The effector domain is conjugated to a targeting domaincomprising a mutant form of factor VII that binds to tissue factor butdoes not initiate blood clotting as described above.

Immunoconjugate proteins of the invention are administered to a patienthaving a disease associated with neovascularization such as cancer,rheumatoid arthritis, the exudative (“wet”) form of maculardegeneration, or atherosclerosis. Administration may be local orsystemic, depending upon the type of pathological condition involved inthe therapy. As used herein, the term “patient” includes both humans orother species; the invention thus has both medical and veterinaryapplications. In veterinary compositions and treatments,immunoconjugates are constructed using targeting and effector domainsderived from the corresponding species.

Administration can be via any method known in the art such as, forexample, intravenous, intramuscular, intratumoral, subcutaneous,intrasynovial, intraocular, intraplaque, or intradermal injection of thepurified immunoconjugate or of a replication-deficient adenoviralvector, or other viral vectors carrying a cDNA encoding a secreted formof the immunoconjugate. Other routes of administration can be parenteraladministration of fluids, and the like. In preferred embodiments, thepatient is treated by intravenous or intratumoral injection, orinjection at other sites, of one or more immunoconjugate proteins, or byintravenous or intratumoral injection, or injection at other sites, ofone or more expression vectors carrying a cDNA encoding a secreted formof one or more types of immunoconjugate proteins. In some embodiments,the patient is treated by intravenous or intratumoral injection of aneffective amount of one or more replication-deficient adenoviralvectors, or one or more adeno-associated vectors carrying cDNA encodinga secreted form of one or more types of immunoconjugate proteins. Amethod employing a replication-deficient adenoviral vector isillustrated hereafter. Many typical embodiments involve intratumoraland/or intramuscular injections of effective amounts of a vectorencoding a secreted form of an immunoconjugate. Where vectors areemployed for cancer, intratumoral injection of the vectors provides animportant safety advantage over intravenous injection, because thevector infects predominantly the cells of the injected tumor.

Administrations involving injections of immunoconjugate proteins employcompositions wherein the immunoconjugate protein, or a combination ofproteins, is dispersed or solubilized in a pharmaceutically acceptablecarrier. In some cases, immunoconjugates are synthesized in DrosophilaS2 cells transfected with the expression vector pMK33/hygromycin, or inChinese hamster ovary (CHO) cells transfected with the expression vectorpcDNA3.1, or in a Baculovirus expression system, each vector carrying acDNA encoding a secreted form of the immunoconjugate.

The amount of immunoconjugate necessary to bring about the therapeutictreatment is not fixed per se, and necessarily is dependent on theconcentration of ingredients in the composition administered inconjunction with a pharmaceutical carrier, adjunct compounds in thecompositions administered that enhance the immune system response morefully illustrated below, and the age, weight, and clinical condition ofthe patient to be treated. Preferred compositions deliverimmunoconjugate(s) in effective amounts without producing unacceptabletoxicity to the patient. Pharmaceutical compositions or formulations ofthe invention may also include other carriers, adjuvants, stabilizers,preservatives, dispersing agents, and other agents conventional in theart having regard to the type of formulation in question.

As applied to cancer, the invention employs immunoconjugates having atargeting domain that specifically targets human tumor cells or tumorvasculature endothelial cells, or both, and an effector domain thatactivates a cytolytic immune response or cytotoxic effect against thetargeted cells. As described above, immunoconjugates that have, as theeffector domain, the Fc region of the IgG1 immunoglobulin, including thehinge, conjugated to mutant human factor VII that does not initiateblood clotting are particularly efficacious in many cancer treatmentsbecause they target tissue factor expressed both on tumor vasculatureand tumor cells. In some embodiments, therapeutic effects can be furtherenhanced by administering to the patient another class ofimmunoconjugates that selectively target the tumor, such asimmunoconjugates that have, as the targeting domain, an anti-human tumorcell scFv or V_(H) antibody fragment isolated by panning a scFv or V_(H)fusion-phage library, derived from the peripheral blood lymphocytes ofcancer patients, against the tumor cells previously described (Cai, X.,and Garen, A. (1997) Proc. Natl. Acad. Sci. USA 94, 9261-9266).Combinations of immunoconjugates, administered simultaneously orsequentially as described above, are particularly advantageous wheresynergy that enhances cytolysis of the targeted cells is observed.

In cancer treatments, anti-tumor immunoconjugates are used for treatinga variety of cancers, particularly primary or metastatic solid tumors,including melanoma, renal, prostate, breast, ovarian, brain,neuroblastoma, head and neck, pancreatic, bladder, and lung cancer. Theimmunoconjugates may be employed to target the tumor vasculature,particularly vascular endothelial cells, and/or tumor cells. The tumorvasculature offers several advantages for immunotherapy, as follows. (i)Some of the vascular targets including tissue factor should be the samefor all tumors. (ii) Immunoconjugates targeted to the vasculature do nothave to infiltrate a tumor mass in order to reach their targets. (iii)Targeting the tumor vasculature should generate an amplified therapeuticresponse, because each blood vessel nourishes numerous tumor cells whoseviability is dependent on the functional integrity of the vessel. (iv)The vasculature is unlikely to develop resistance to an immunoconjugate,because that would require modification of the entire endothelium layerlining a vessel. Unlike previously described antiangiogenic methods thatinhibit new vascular growth, immunoconjugates of the invention elicit acytolytic response to the neovasculature.

Immunoconjugates to the invention can also be effective for treatingpatients with rheumatoid arthritis, the exudative (“wet”) form ofmacular degeneration, atherosclerosis, and other diseases associatedwith neovascularization. Administering an immunoconjugate targeted totissue factor by a mutated human factor VII, which is conjugated to theFc domain of an IgG1 immunoglobulin, can generate a cytolytic immuneresponse against the vascular endothelial cells that invade the synoviumin rheumatoid arthritis and express tissue factor. Likewise, factor VIIimmunoconjugates can also be effective for treating the exudative (wet)form of macular degeneration because of the extensive neovascularizationobserved in that pathologic condition. Immunoconjugates of the inventioncan also be effective for the treatment of atherosclerosis by generatinga cytolytic immune response against cells expressing tissue factor inplaques.

In summary, an overall immunotherapy program according to the inventioninvolves constructing immunoconjugates containing, as the targetingdomain, factor VII with a mutated active site, which binds with highavidity and specificity to tissue factor expressed on neovascularendothelial cells and also on tumor cells without causing bloodcoagulation. Immunoconjugates that contain as the effector domain the Fcregion of an IgG1 immunoglobulin mediate the lysis of targeted cells byNK cells and complement, resulting in destruction of the neovasculature.A significant advantage of the invention is the fact thatimmunoconjugates containing two factor VII targeting domains, incontrast to endogenous factor VII monomeric molecules, exhibitsignificantly greater binding than exogenous factor VII to cellsexpressing tissue factor, successfully competing in the presence ofexcess natural ligand. Example 2 below illustrates an immunoconjugatethat successfully competed in the presence of at least about a ten-foldmolar excess of natural ligand, but the invention encompasses otherembodiments exhibiting lower or higher affinity. The binding betweenendogenous factor VII to tissue factor is one of the most specific andstrongest reactions known, with a K_(d) in the picomolar range.Nevertheless, this invention improves significantly on the normalbinding of factor VII to tissue factor as a result of the avidity effectof having two factor VII sequences in one immunoconjugate molecule. Thisavidity effect enhances the targeting and binding of immunoconjugates ofthe invention, so that they compete effectively with endogenous factorVII and the binding persists longer, so that the immune responseelicited is maximized. The immune response may be further maximized byadministering to a patient, as adjunct therapy, another immunoconjugatehaving an effector domain which is the Fc region of an IgG1immunoglobulin conjugated to a targeting domain which is a scFv or V_(H)antibody fragment that binds to neovasculature, and/or, in the case ofcancer, to the patient's type of tumor cell.

EXAMPLES

The examples presented herein further illustrate and explain the presentinvention and should not be taken as limiting in any regard. Some of themethods used to generate and characterize immunoconjugates of theinvention and component monoclonal antibodies have ben described in Cai,X., and Garen, A. (1995) Proc Natl. Acad. Sci. USA 92, 6537-6541, (1996)Proc. Natl. Acad. Sci. USA 93, 6280-6285, and the 1997 reference citedabove; in PCT/IB96/01032 to Yale University and Garen and Cai, published23 Jan. 1997 as WO 97/02479, presently pending in the U.S. asapplication Ser. No. 08/983,607 filed Apr. 27, 1998, allowed Mar. 3,2000; in Wang, et al., cited above; and in Hu, Z., et al., cited above.

Example 1

Immunoconjugates containing a human single-chain Fv (scFv) targetingdomain conjugated to the Fc effector domain of human IgG1 aresynthesized and tested in this example. The scFv targeting domains wereoriginally isolated as melanoma-specific clones from a scFv fusion-phagelibrary, derived from the antibody repertoire of a vaccinated melanomapatient (more fully described in the Cai and Garen references citedabove). The purified immunoconjugates showed similar binding specificityas did the fusion-phage clones: Binding occurred to human melanoma cellsbut not to human melanocytes or to several other types of normal cellsand tumor cells.

Materials and Methods. Cell cultures. The permanent human melanoma linesA2058 (American Type Culture Collection, Rockville, Md.) and TF2 weregrown in DMEM+10% FCS. Primary cultures of human microvascularendothelial cells and fibroblast cells were extruded from newbornforeskin and cultured in RPMI medium supplemented with 8% FBS and 2%human peripartum serum; the endothelial cells were further supplementedwith 33 mM 3-isobutyl-1-methylxanthene (IBMX) and 0.5 mM dibutyryl cAMP.Primary cultures of human melanocytes from newborn foreskin wereprepared by the Skin Disease Research Center at Yale University Schoolof Medicine. The transformed human kidney cells 293-EBNA (Invitrogen)and the Chinese hamster ovary (CHO) cells were grown in RMPI+10% FCS.(v) Drosophila cells (Schneider S2) were grown at 25° C. in Ex-cell 301medium (JRH biosciences)+10% FBS. Resting NK cells were isolated fromnormal donors by leukophoresis and immunoselection and were used within18 hr after isolation; most of the cells (>97%) were CD3−, CD56+ andCD16+.

Preparation of the immunoconjugates. The procedures involvedtransfecting the expression vector pcDNA3.1 (Invitrogen) into CHO cells,or the expression vector pMK33/pMtHy into Drosophila cells; each vectorcarried a cDNA encoding a secreted immunoconjugate (FIG. 1). The cDNAsfor the scFv targeting domains were synthesized from the correspondingfusion-phage (1) using PCR primers containing SacI or BamHI sites as,follows: a) GTCGAGCAGAGCTCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGTGAAGAAGCC(SEQ ID NO: 1); b) ACGTTCAGGGGATCCACCTAGGACGGTCAGCTTGGTCCC (SEQ ID NO:2). The human Fc effector domain was synthesized from a cDNA libraryderived from human peripheral blood lymphocytes, using PCR with primerscontaining BamHI and SalI sites, as follows: a)ACCTTGCAGGATCCGCAAGACCCAAATCTTGTGACCAAAACTCAC (SEQ ID N: 3); b)GATCACGTGTCGACTTATCATTTACCCGGAG ACAGGGAGAGGCTCTTCTG (SEQ ID NO: 4). ThecDNA for the IgG1 leader was synthesized by hybridizing twocomplementary oligonucleotides containing EcoRI and SacI ends, asfollows: a)AATTCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTTGCTGCATTAAGAGGTGTCCAGTCCGAGCT (SEQID NO: 5); b) CGGACTGGACACCTGTTA ATGCAGCAACAAGAAAAGCCAGCTCAGCCCAAACTCATG(SEQ ID NO: 6).

These three cDNAs encoding a secreted immunoconjugate were ligated firstinto a cloning vector for sequencing and then into the expressionvectors pcDNA3.1 and pMK33/pMtHy for transfection into CHO or DrosophilaS2 cells, respectively. The transfection procedure for CHO cellsinvolved growing the cells in RPMI+10% FCS and transfecting with 5 μg ofan expression vector using Superfect™ (Qiagen). Stable transfectantswere selected in RPMI+10% FCS+1 mg/ml G418. For protein expression,transfected CHO cells were first adapted to growth in CHO serum-freemedium (Sigma), and then were grown for 3 days as a suspension culture(2×10⁵ cells/ml) in the serum-free medium. The transfection procedurefor Drosophila S2 cells involved growing the cells in Ex-cell medium+10%FBS and transfecting with 10 μg of an expression vector usingLipofectin™ (Gibco-BRL). Stable transfectants were selected in Ex-cellmedium+10% FBS+300 μg/ml hygromycin, and adapted for growth as asuspension culture in serum-free Ex-cell medium. Expression of theencoded immunoconjugate was induced in the suspension culture byaddition of 500 μM copper sulfate.

The immunoconjugates secreted by transfected CHO or Drosophila S2 cellswere purified from the culture medium by affinity chromatography on aprotein-A matrix (Pierce).

Binding specificity of the immunoconjugates measured byfluorescence-activated cell sorting (FACS). Melanoma cells and controlcells were harvested in non-enzymatic dissociation medium (Sigma),washed with PBS/BSA+0.1% sodium azide, and incubated in PBS/BSA withadded immunoconjugate (1 μg/ml) or in PBS/BSA without an immunoconjugateas a control. The cells were washed with PBS/BSA, incubated 30 min at 4°C. with fluoroscein-labeled anti-human Fc γ-chain (Vector), and analyzedon a Becton-Dickenson FACsort instrument.

Immunoprecipitation of a melanoma cell extract with an immunoconjugate.A sample of about 1×10⁷ cells from the human melanoma line A2058 wassuspended in a solution containing 10 μg/ml immunoconjugate, 1% BSA and0.05% sodium azide in PBS, and was incubated for 30 minutes on ice. Thecells were washed twice with PBS and lysed in a solution containing 1%NP-40, 1 μg/ml immunoconjugate and 0.2 mM PMSF in PBS for 20 minutes onice. The lysate was spun at 13,000 RPM in microfuge for 5 min, and thesupernate was recovered and incubated with protein-G beads overnight ona rotator. The beads were collected, washed twice with a solutioncontaining 1% NP-40 in PBS and once with PBS, and the beads werecollected, boiled in PAGE loading buffer and analyzed by PAGE.

Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS)and Liquid Chromatography Tandem MS (LC/MS/MS) protein identification. Aprotein band stained with Coomassie Blue was excised from the gel anddigested with trypsin as described athttp://info.med.yale.edu/wmkeck/geldig3.htm. A sample of the trypticdigest was analyzed by MALDI-MS on a Micromass TofSpec SE. To attain thehigh level of accuracy needed for peptide mass searching, 100 fmolbradykinin which has a protonated monoisotpic mass of 1060.57, and ACTHclip which has a protonated monoisotopic mass of 2465.2, were used asinternal calibrants. The resulting monoisotopic masses of the trypticpeptides were searched against the OWL database with the ProFoundprogram using a mass tolerance of 0.2 daltons, and against theEMBL/non-redundant database with the PeptideSearch program using a0.015% mass tolerance. Other important criteria used in the search werea mass range that extended from 140-560 Kda, a maximum of 1 missedcleavage and no limitation with regard to taxonomy. All the proteinchemistry and mass spectronomy studies were carried out in the W. M.Keck Foundation & HHMI Biopolymer Laboratory at Yale University. Furtherinformation can be found at http://info.med.yale.edu/wmkekc/.

A sample of the trypsin-digested protein band used for the MALDI-MSanalysis was also analyzed on a LCQ ion trap mass spectrometer. ASequest search of the MS/MS data was done, using a tandem masscorrelation algorithm with a mass toerance of 2.0 daltons, to determinewhether significant similarities exist between peptides from the trypticdigest and the reconstructed theoretical spectra for a protein in theNCBI or database. Further information about this procedure can beobtained at http://info.med.yale.edu/wmkeck/prochem.htm#ms/mspi.

Assays for immunoconjugate-dependent cytolysis of human melanoma cellsby NK cells and complement. The Calcein-AM retention procedure was usedfor both assays. The target cells, either human melanoma lines orprimary human fibroblast cells, were isolated from culture flasks in anon-enzymatic dissociation medium (Sigma) and added to 96-well plates(2×10⁴ cells/well). The adherent target cells were washed once with PBS,and afterwards incubated for 20 minutes at 37° C. in serum-free culturemedium (GIBCO-BRL) containing an immunoconjugate (1 μg/ml) or without animmunoconjugate as a control. The target cells were then labeled with 7μM Calcein-AM (Molecular Probes Inc., Eugene, Oreg.) in serum-freemedium for 40 min at 37° C. Calcein-AM is a fluorescent dye that entersthe cells, where it is enzymatically altered and remains intracellularuntil the cells are lysed. For the cytolytic assays involving NK cells,the labeled target cells were incubated 3-4 hr at 37° C. with human NKcells using the indicated ratios of NK cells to target cells. For thecytolytic assays involving complement, the labeled target cells wereincubated 1 hr at 37° C. with human serum or purified rabbit complementcomponents (Cedarlane Laboratories, Ontario, Canada). After incubationwith NK cells or complement, the target cells were washed twice withPBS, and the fluorescence in the remaining adherent cells (residualfluorescence) was measured with a plate reader. The maximum attainablecytolysis of the target cells was determined by measuring residualfluorescence of the target cells after treatment with lysis buffer (50mM sodium borate, 0.1% Triton X-100, pH 9.0) for 3 hr at 37° C. Themaximum residual fluorescence was determined by measuring thefluorescence of adherent target cells that were not exposed to NK cellsor complement. The % cytolysis for each sample of target cells wascalculated as follows: (residual fluorescence of the sample target cellsminus residual fluorescence of the lysed target cells)/(maximum residualfluorescence of the target cells minus residual fluorescence of thelysed target cells).

Results. Synthesis and characterization of the anti-melanomaimmunoconjugates. The immunoconjugate encoded a human IgG1 leader forsecretion, a human scFv domain for targeting melanoma cells, and a humanIgG1-Fc effector domain (FIG. 1). The immunoconjugate molecules wereexpressed in transfected CHO and Drosophila S2 cells, and were purifiedfrom the culture medium as two-chain molecules linked by disulfidebridges in the hinge region of the Fc domain (FIG. 1). Twoimmunoconjugates were synthesized, each containing a scFv targetingdomain derived from the fusion-phage clone E26-1 or G71-1. The bindingspecificities of the immunoconjugates E26-1 and G71-1 were tested bycell sorting (FACS) using two human melanoma lines; the controls wereprimary cultures of normal human melanocyte, fibroblast, microvascularand umbilical vascular endothelial cells, and the human kidney line293-EBNA. The results showed that the immunoconjugates bind strongly tothe melanoma cell lines but do not bind to the controls, consistent withthe results obtained with the E26-1 and G71-1 fusion-phage clones. Thebinding to melanoma cells occurred when the cells were collected fromthe culture flask using a non-enzymatic cell dissociation medium(Sigma), but not when the dissociation medium contained trypsin or whenthe dissociated cells were subsequently treated with trypsin. Thisfinding indicated that the cognate melanoma antigen(s) for theimmunoconjugates is located on the surface of melanoma cells. Theantigen(s) appear to be exceptionally sensitive to trypsin, since thesame exposure to trypsin did not affect the binding to the melanomacells of antibodies against the cell surface molecules ICAM-1, MHCclass-I and tissue factor.

Identification of the cognate melanoma antigen for the immunoconjugates.Cultured cells from the human melanoma line A2058 were equilibrated withthe immunoconjugates G71-1 or E26-1, and the cells were lysed withdetergent. The immunoconjugate-antigen complex in the lysate wascollected on protein-G beads and analyzed by PAGE. A protein band withan apparent molecular weight of 250 kda was detected in the melanomacells but not in the control. Analysis of a tryptic digest of theprotein band by the MALDI-MS procedure identified 75 peptide masses thatwere not present in a digest of a control gel slice. A search of proteinsequence databases by ProFound yielded 50 peptide masses that matchedpeptide masses in the MCSP core protein, spanning 26% of the completeprotein sequence. The ProFound probability score for this identificationwas 1.0, and the next closest score was 2.2E-61. A search by PeptideSearch matched 45 peptides to the MCSP core protein, with 38 peptidesrepresenting the next closest match.

The tryptic digest of the 250 kda protein was also analyzed by theLC/MS/MS procedure (Stone, K. L., et al. (1998) Electrophoresis 19,1046-1052). A Sequest search of the MS/MS data from the tryptic digestshowed significant similarity between the MS/MS spectra for two or morepeptides and the reconstructed theoretical MS/MS spectra for two or morepeptides from the MCSP core protein in the NCBI nr database.

The results of both mass spectroscopy analyses indicate that themelanoma protein immunoprecipitated by the immunoconjugates G71-1 andE26-1 matched the MCSP core protein.

Immunoconjugate-dependent cytolysis of melanoma cells mediated by NKcells and complement. One of the cytolytic pathways of the immune systeminvolves NK cells which can bind directly to target cells, causingantibody-independent lysis of the target cells. NK cells also bind tothe Fc effector domain of an antibody, resulting in anti-body-dependentlysis of cells that bind to the targeting domain of the antibody. Thisantibody-dependent cell-mediated cytolytic pathway (ADCC) should alsocause lysis of cells that bind to the targeting domain ofimmunoconjugates containing an Fc effector domain. To test for an ADCCresponse dependent on the immunoconjugates E26-1 and G71-1, melanomacells and fibroblast control cells were labeled with the fluorescent dyeCalcein-AM, and the labeled cells were incubated with human NK cellsalone or together with an immunoconjugate. Cytolysis was assayed bymeasuring the amount of fluorescent dye retained in the cells thatremained intact. The results for E26-1 (FIG. 2) show that afterincubation with the immunoconjugate and NK cells, the percentage oflysed melanoma cells increased above the basal level that occurs withoutthe immunoconjugate, reaching almost 100% lysis at a 20/1 ratio of NKcells to melanoma cells. In contrast to the efficient lysis of melanomacells, the fibroblast cells showed no significant increase in cell lysisafter incubation with the immunoconjugate and NK cells. Similar resultswere obtained with the G71-1 immunoconjugate.

The sensitivity of target cells to lysis by NK cells is increased byexpression on the target cell surface of adhesion molecules such asICAMs, and is reduced by expression of MHC class I molecules (Zami, L.,et al. (1995) Cell. Immunol. 164, 100-104 and Storkus, W. J., et al.(1989) Proc. Natl. Acad. Sci. USA 86, 2361-2364). To determine whetherdifferences in expression of these molecules might contribute to thedifferences in the sensitivities of melanoma cells and fibroblast cellsin the ADCC assays, the expression of ICAM-1 and MHC class I moleculesby melanoma and fibroblast cells was measured by FACS. Expression ofboth molecules was similar in the two cell types, indicating that thespecific lysis of melanoma cells by NK cells depends on the binding ofthe immunoconjugates to the cognate antigens expressed on melanomacells.

Another cytolytic pathway of the immune system involves the complementcascade, which is activated when the molecule C1q reacts with the Fcregion of antibodies bound to a target cell (Bruggemann M., et al.(1987) J. Exp. Med. 166, 1351-1361). To test for a complement-mediatedcytolytic response against melanoma cells, dependent on theimmunoconjugates E26-1 and G71-1, the same assay procedure employed forthe NK-mediated cytolytic response reported above was used with onechange, namely that human serum or rabbit serum, which contain thecomponents of the complement cascade, was substituted for NK cells. Theresults (FIG. 3 and Table 1) show that after incubation with theimmunoconjugates and either human serum or rabbit serum, there was anincrease in the fraction of melanoma cells lysed from 4% to almost 100%.In contrast to the efficient lysis of melanoma cells, the fibroblastcells showed a small increase in the fraction of lysed cells afterincubation with the immunoconjugates and human serum, and no significantincrease after incubation with the immunoconjugates and rabbit serum.

TABLE 1 Immunoconjugate and complement reagents used for FIG. 3 assays.Human Rabbit Immunoconjugate Assay serum complement G71-1 E26-1 A, F − +− − B, G + − + − C, H − + + − D, I + − − + E, J − + − +

Discussion. For the present study the scFv molecules from two of theclones were used as the targeting domains for constructingimmunoconjugates containing a human IgG1-Fc effector domain (FIG. 1).The protein immuno-precipitated from human melanoma cells by bothimmunoconjugates was identified by mass spectroscopic analyses as thecore protein of a melanoma-associated chondroitin sulfate proteoglycan(MCSP) (Bumol, T. F. & Reisfeld, R. A. (1982) Proc. Natl. Acad. Sci. USA79, 1245-1249 and Bumol, T., et al. (1984) J. Biol. Chem. 259,12733-12741). The MCSP molecule was first identified as the cognateantigen recognized by the mAb 9.2.27, and it appears to be the cognateantigen for several other mAbs (Reisfeld, R. A. & Cheresh, D. A. (1987)Adv. Immunol. 40, 323-377; Wilson, B. S., et al. (1981) Int. J. Cancer28, 293-300; Hellstrøm, I., et al. (1983) J. Immunol. 130, 1467-1472).Several scFv fusion-phage clones that bind to the melanoma antigenHMW-MAA, which probably is the same as MCSP, have been isolated from asynthetic human scFv library by panning against purified HMW-MAA (Desai,S. A., et al. (1998) Cancer Res. 58, 2417-2425). The MCSP molecule isexpressed predominantly on the surface of most human melanoma cells, andalso on capillary endothelial cells of glial tumors. The finding thatMCSP is the cognate antigen for at least two of the melanoma-specificclones isolated from a melanoma patient's scFv fusion-phage library bypanning against melanoma cells, suggests that MCSP is a dominantmelanoma antigen in vivo.

As an initial test of the therapeutic potential of the twoimmunoconjugates, an in vitro cytotolytic assay involvingfluorescent-labeled target cells was used to determine the capacity ofthe immunoconjugates to target human melanoma cells for lysis by NKcells and by complement. Both of the immunoconjugates produced a sharpincrease in the cytolytic activity of NK cells and complement againstmelanoma cells, resulting in virtually complete lysis of the targetedmelanoma cell population and only minor or no increase in lysis of thefibroblast cells used as a control. There was also a significantbackground of immunoconjugate-independent cytolysis of melanoma cellsand fibroblast cells by NK cells and complement, which is expected foran allogenic assay in which the tumor cells, NK cells and complement areisolated from different individuals (Ciccone, E., et al. (1992) J. Exp.Med. 175, 709-718). This background should be reduced in a cancerpatient, because all of these components are autologous.

The results of the in vitro cytolytic tests provide preliminary evidencethat immunoconjugates could have a potential role in immunotherapyprotocols with melanoma and other cancers for which tumor-specific scFvor VH targeting domains are available. A limited phase-I clinical trialfor melanoma immunotherapy with the mouse mAb 9.2.27, which binds toMCSP, showed specific localization of the antibody in the tumors withoutevidence of associated toxicity (Oldham, R. K., et al. (1984) J. Clin.Oncol. 2, 1235-1244). The scFv immunoconjugates that bind to MCSP shouldbe more effective than a mouse mAb for immunotherapy, because thesmaller molecule size should improve tumor penetration, and the humanderivation of the molecule should minimize an immune rejection response.The G71-1 and E26-1 immunoconjugates probably bind to different MCSPepitopes, as suggested by major differences in their V_(H) sequences(see SEQ ID NO: 11 and SEQ ID NO: 12 and the next example), andtherefore could be administered together to enhance therapeuticefficacy.

Example 2

This example reports that an immunotherapy treatment for cancer thattargets both the tumor vasculature and tumor cells has shown promisingresults in a severe combined immunodeficient mouse xenograft model ofhuman melanoma. The procedure involved systemic delivery to SCID mice oftwo immunoconjugates, each composed of a tumor-targeting domainconjugated to the Fc effector domain region of a human IgG1. Theeffector domain induces a cytolytic immune response against the targetedcells by natural killer (NK) cells and complement. The immunoconjugatesare encoded as secreted molecules in a replication-incompetentadenoviral vector.

Materials and Methods. Cell lines. The melanoma cell lines LXSN, TF2 andLXSN/VEGF were derived from the human melanoma line YU-SIT1 byretroviral-mediated transfection and cloning. The LXSN line wastransfected with the control retrovirus and expresses a low level of TF.The TF2 line was transfected with a retrovirus encoding TF cDNA andexpresses a high level of TF. The LXSN/VEGF line was transfected with aretrovirus encoding VEGF cDNA and expresses high level of VEGF. Thehuman kidney line 293 was purchased from the American Type CultureCollection.

Plasmid vector. The construction of the plasmid vector encoding the scFv(G71-1) immunoconjugate was described in Example 1 (see also SEQ ID NO:11). For the construction of the vector encoding the mouse factor VII(mfVII) immunoconjugate, the mfVII cDNA was amplified by PCR from amouse liver cDNA library (Quick-Clone cDNA, Clonetech) using the5′-primer ACGATCTTAAGCTTCCCCACAGTCTCATCATGGTTCCA (SEQ ID NO: 7) and the3′-primer ACGGTAACGGATCCCAGTAGTGGGAGTCGGAAAACCCC (SEQ ID NO: 8). Theamplified mfVII cDNA, which contains the leader and coding sequenceswithout a stop codon, was cloned into the HindIII and BamHI sites of thepcDNA3.1 (+) vector (Invitrogen) in-frame with a cDNA encoding the humanIgG1 Fc domain. The vector DNA was amplified in HB101 competent cells(Life Technologies) and sequenced. The active site of mfVII cDNA wasmutated by substituting an alanine codon for lysine-341 (Dickinson, C.D., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 14379-14384). Themutagenesis procedure was done as described in the QuickChangesite-directed mutagenesis manual (Stratagene). The 5′-primer wasGGTACCAAGGACGCCTGCGCGGGTGACAGCGGTGGCCCA (SEQ ID NO: 9) and the 3′-primerwas TGGGCCACCGCTGTCACCCGCGCAGGCGTCCTTGGTACC (SEQ ID NO: 10). The mfVIIcDNA with the active site mutation is designated mfVIIasm. The plasmidcontaining mfVIIasm cDNA was transformed into HB101 competent cells, andtransformed colonies were selected on 2×TY/carbenicillin agar. Thesequence of the plasmid DNA showed a substition of an alanine codon(GCG) for the lys-341 codon (AAG) in the mfVIIasm DNA.

Synthesis of immunoconjugates in CHO cells. The procedures fortransfecting the immunoconjugate cDNAs into CHO cells and isolatingclones were described in Example 1. The transfected CHO cells werecultured in CHO serum-free medium (EX-CELL 301, JRH Biosciences); forsynthesis of the mfVIIasm immunoconjugate, the CHO serum-free medium wassupplemented with vitamin K1 (Sigma) to a final concentration of 1μg/ml. The immunoconjugates were purified by affinity chromatography onProtein A beads (Pierce) and were concentrated and desalted bycentrifugation through an Ultrafree-15 Biomax-50 filter (Millipore) andadjusted to 10 mM Tris-HCl pH 8.0. The immunoconjugate concentrationswere measured by the Bio-Rad protein assay procedure.

Fluorescence-activated cell sorting (FACS). Melanoma cells wereharvested in nonenzymatic dissociation solution (Sigma), washed andresuspended in TBS/BSA/Ca²⁺ (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 20 mMCaCl₂, 1% BSA and 0.1% NaN₃). An immunoconjugate was added (5 μg/mlfinal concentration) and the cells were incubated for 30 min either at37° C. for the mfVIIasm immunoconjugate or on ice for the G71- 1immunoconjugate; the control cells were incubated without addedimmunoconjugate. After incubation the cells were washed with TBS/BSA,incubated 30 min on ice with fluorescein-labeled anti-human Fc γ-chain(Vector Laboratories), and analyzed on a Becton-Dickenson FACsortinstrument.

Adenoviral vectors: The adenoviral vector system consists of the shuttlevectors pAdTrack-CMV and pShuttle-CMV, and the backbone vector pAdEasy-1(He, T. C., et al. (1998) Proc. Natl. Acad. Sci. USA 95, 2509-2514). Theimmunoconjugate cDNAs were isolated from the pcDNA3.1 plasmid vectors bydigestion with HindIII followed by Klenow fragment to fill-in the3′-recessed end, and then were digested with NotI to release the cDNAinsert which was purified by agarose gel electrophoresis. The shuttlevectors were first digested with Kpn1 followed by Klenow fragment, andthen were digested with Not1. The immunoconjugate cDNAs were ligatedinto the shuttle vectors by incubation with T4 DNA ligase at 16° C.overnight, and the shuttle vectors were transformed into HB101 competentcells by heat-shock. Transformed colonies were selected on2×TY/kanamycin agar, and the shuttle vectors were extracted andpurified. The purified shuttle vectors and pAdTrack-CMV DNAs weredigested with Pme1 at 37° C. for 2 hr. A mixture of 500 ng shuttlevector DNA and 100 ng pAdEasy-1 DNA was electroporated into BJ5183competent cells, and the cells were shaken at 37° C. for 15 min andplated on LB/kanamycin agar. The plates were incubated at 37° C.overnight, and transformed colonies were isolated. The plasmid DNAs werepurified by minipreps and screened for recombinant adenoviral DNA byelectrophoresis on 0.6% agarose gels.

The recombinant adenoviral DNAs encoding the immunoconjugates weretransfected into 1×10⁵ 293 cells, following the protocol described abovefor transfecting CHO cells. The cells were collected 7 days aftertransfection, and the adenoviruses were released by 3 freeze-thaw cyclesand amplified by infecting 293 cells in one 150 mm culture plate. After2 days the adenoviruses were harvested as described above and amplifiedagain by infecting 293 cells in 20 culture plates. The amplifiedadenoviruses were harvested 2 days later and purified by centrifugationin CsCl. The final yields usually were about 10¹³ virus particles asestimated from the absorbance at 260 nm; the conversion is 1 O.D.unit=1×10¹² particles. The purified adenoviruses were dialyzed againstPBS and stored at −80° C.

SCID mice experiments. The SCID mice were 4- to 5-weeks old females fromTaconic laboratories. The mice were injected subcutaneously into theright rear flank with 5×10⁵ TF2 or LXSN human melanoma cells. After thetumors had grown to a palpable size below the skin surface (˜5 mm³) orto a larger size above the skin surface (˜50 mm³), the mice wereinjected via the tail vein with the adenoviral vector encoding animmunoconjugate, or as a control with the adenoviral vector that doesnot encode an immunoconjugate. The concentration of immunoconjugateprotein secreted into blood was measured by collecting about 0.1 ml ofblood from one eye into a microcapillary tube coated with heparin, andcentrifuging the blood to remove cells. The supernatant plasma wasdiluted with sodium bicarbonate buffer pH 9.6 and distributed into wellsof pro-bind assay plates (Falcon), and the plates were incubated firstat 37° C. for 2 hr and then at 4° C. overnight. The wells were blockedwith 5% nonfat milk in PBS for 30 min and washed 3 times with PBS, and aperoxidase-labeled anti-human IgG antibody diluted 1:2000 in 5% nonfatmilk was added to the wells. The plates were incubated for 1 hr at roomtemperature and washed in PBS, and the peroxidase substrate OPD wasadded and absorbance was measured at 490 nm in a microplate reader. Theprotein standard was human IgG (Sigma) purified by chromatography onProtein A beads.

The size of a tumor appearing on the skin of a SCID mouse was measuredin two dimensions with a caliper, and the tumor volume was estimated bythe formula (width)² (length)/2. At the end of an experiment, the micewere dissected and the tumors were weighed. The organs were examined formorphological evidence of damage, and paraffin sections were preparedfor histological examination.

Immunohistochemistry. Paraffin sections of the tumors and organs wereincubated in PBS+0.3% H₂O₂ for 30 min and blocked in TBS/BSA buffer for30 min. A solution containing 10 μg/ml of the mfVIIasm immunoconjugatein TBS/BSA/Ca²⁺ buffer, or as a control the buffer without theimmunoconjugate, was added to the sections and incubated at 37° C. for 1hr. After washing 3 times in the same buffer, the sections wereincubated at room temperature for 1 hr with anti-human γ-chain antibodylabeled with alkaline phosphatase, stained with BCIP/NBT which producesa blue color, and counterstained with methyl green.

Results. Properties of the immunoconjugates. The scFv (G71-1) and themutated mouse factor VII (mfVIIasm) immunoconjugates were synthesized inCHO cells and purified from the culture medium by affinitychromatography on Protein A beads. An earlier analysis by SDS-PAGEshowed that the G71-1 immunoconjugate is composed of two identicalchains, presumably coupled by disulfide bridges between the hingeregions of the Fc domains (Example 1). The same result was obtained withthe mfVIIasm immunoconjugate has two targeting domains, as compared tothe single targeting domain in the monomeric endogenous fVII molecule,it can bind cooperatively to two TF molecules, resulting in strongerbinding than endogenous fVII to cells expressing TF. A competitive FACSassayed showed that human fVIIa competes on an equimolar basis with themfVIIasm immunoconjugate for binding to half of the accessible sites onhuman melanoma cells, probably because only one of the targeting domainson the immunoconjugate molecule can bind to TF at these sites. Thebinding of the mfVIIasm immunoconjugate to the remaining sites could notbe competed in the presence of a tenfold excess of human fVIIa,suggesting that both targeting domains of the immunoconjugate moleculecan bind at these sites and provide a strong avidity effect. It appearsthat only about half of the TF molecules on the melanoma cells aresufficiently close to a second TF molecule to form a cooperative bindingsite for both targeting domains on a mfVIIasm immunoconjugate.

The xenografts for the immunotherapy tests were generated from the humanmelanoma lines LXSN and TF2 which express, respectively, low or highlevels of TF. The mfVIIasm immunoconjugate binds more extensively to theTF2 cells than to the LXSN cells as determined by FACS, consistent withthe higher level of TF expression by TF2 cells. The mfVIIasmimmunoconjugate was also tested by immunohistochemistry for binding tosections of a human melanoma xenograft generated from the melanoma lineLXSN/VEGF which produces a high level of VEGF, resulting in a denselyvascularized xenograft. Binding occurred to the tumor vascularendothelial cells as well as to the tumor cells (FIG. 4), indicatingthat TF is expressed by both cell types in the xenograft.Immunohistochemistry tests with sections of normal mouse liver, kidney,lung and brain showed that the mfVIIasm immunoconjugate does not bind tovascular endothelial cells in these tissues, consistent with otherevidence that TF is not expressed by vascular endothelial cells ofnontumorous tissues.

Immunotherapy tests. For systemic delivery to SCID mice, eachimmunoconjugate was encoded as a secreted molecule in thereplication-defective adenoviral vector system based on pAdEasy-1 (He,et al., cited above), and the vectors were injected into the tail veinof mice that had first been injected subcutaneously with human melanomacells. The initial immunotherapy tests involved injecting each vectorseparately, and both vectors together, into the mice that had developeda palpable TF2 xenograft. A total of three injections were administeredat weekly intervals, and the experiment was terminated 6 days after thelast injection. The concentration of the immunoconjugates in the bloodwas monitored by ELISA after the first and second injections (FIG. 5).The average concentration after the first injection was 4 mg/ml for theG71-1 immunoconjugate and 0.04 mg/ml for the mfVIIasm immunoconjugate,indicating that the rate of synthesis was about 100-fold higher for theG71-1 immunoconjugate than for the mfVIIasm immunoconjugate. Theconcentration of each immunoconjugate increased after the secondinjections, indicating that additional cells had been infected by theadenoviruses. The growth of the xenografts was monitored by measuring intwo dimensions the size of the tumor appearing on the skin surface, andusing the measurements to estimate the tumor volume (FIG. 6). In thecontrol mice injected with the adenovirus that does not encode animmunoconjugate, the tumor grew continuously at a relatively fast rate,reaching an average volume of about 2,000 mm³ after 20 days. In the miceinjected with an adenovirus encoding an immunoconjugate, tumor growthwas inhibited; the inhibition was stronger for the mfVIIasmimmunoconjugate than for the G71-1 immunoconjugate. All of the miceremained active and appeared healthy at the end of the experiment, andhistological examination of the liver, spleen, lung, kidney and braindid not show any evidence of necrosis, clotting or bleeding. The tumorweights after autopsy were lower in the mice treated with theimmunoconjugates than in the control mice, consistent with the estimatedtumor volumes (FIG. 7). The strongest reduction of tumor weight occurredin the mice treated with both immunoconjugates.

The next two experiments were designed to test two parameters that couldaffect the therapeutic efficacy of the immunoconjugates, namely theinitial size of the xenograft and the level of TF expression by themelanoma cells. (i) The preceding immunotherapy tests involved palpablemelanoma xenografts that had grown to an estimated volume of about 5mm³, corresponding to a small tumor in humans. To test the therapeuticefficacy of the immunoconjugates against a larger xenograft, TF2xenografts were allowed to grow to an estimated volume of about 50 mm³before starting tail vein injections of the two adenoviral vectors. Themice received 4 injections during a period of 3 weeks, and theexperiment was terminated 2 days after the last injection. The averagetumor volume in the mice injected with the adenoviruses encoding theimmunoconjugates was about the same at the end as at the start of theexperiment, in contrast to the average tumor volume in the mice injectedwith the control adenovirus which increased by a factor of about 27during the same period (FIG. 8). These results show that tumor growth isinhibited as effectively with the larger tumor as with the smallertumor. One of the 5 mice injected with the adenovirus encoding theimmunoconjugates died 5 days after the third injection; the cause ofdeath could not be determined because the mouse was not recovered intime for examination. (ii) A parameter that might affect the efficacy ofthe mfVIIasm immunoconjugate is the level of TF expression, which variesamong different tumors (Callender, N. S., et al. (1992) Cancer 79,1194-1201). To study the effect of varying the expression of TF by themelanoma cells in a xenograft, the melanoma line LXSN was used togenerate a xenograft expressing a low level of TF, for comparison withthe xenograft generated from the related line TF2 which expresses ahigher level of TF (Bromberg, M. E., et al. (1999) Proc. Natl. Acad.Sci. USA 92, 8205-8209). After the xenografts reached a palpable size,the mice received during the next 3 weeks 5 injections of the adenovirusencoding the mfVIIasm immunoconjugate or the control adenovirus (FIG.11). In the 5 mice injected with the control adenovirus the xenograftgrew continuously, the average volume increasing to 1350 mm³ on thesecond day after the last injection. During the same period the averagevolume of the xenografts in the mice injected with the mfVIIasmimmunoconjugate increased to 20 mm³, indicating that the inhibition oftumor development is comparable for the LXSN and TF2 xenografts (compareFIGS. 9 and 6). The autopsies performed one day after the last injectionshowed that the xenograft had been eradicated in 2 of the 5 miceinjected with the adenovirus encoding the mfVIIasm immunoconjugate; theaverage tumor weight in the other 3 mice was 0.11 gm as compared to theaverage weight of 0.75 gm in the 5 mice injected with the controladenovirus. The small tumors recovered from these 3 mice showedextensive regions of cell necrosis, which did not occur in the largertumors from the control mice (FIG. 10). All of the mice appeared healthyat the end of this experiment, but a morphological examination of thedissected mice revealed damage to the liver and spleen in the 5 miceinjected with the adenovirus encoding the mfVIIasm immunoconjugate.Histological examination of the liver and spleen showed that many of theliver cells were enlarged and the spleen was extensively infiltratedwith erythrocytes. Enlarged liver cells also occurred in a previousexperiment after 3 injections of the adenovirus encoding the mfVIIasmimmunoconjugate, but the spleen was normal, indicating that the defectsin the spleen developed in the course of the last 2 injections. One ofthe mice also had a subdural brain hemorrhage, which did not occur inother mice from this experiment or any of the previous experiments. Itis uncertain whether this defect was induced by the binding of themfVIIasm immunoconjugate to TF expressed in the brain vasculature, oroccurred spontaneously.

Discussion. The immunotherapy procedure in this example involvedsystemic delivery to SCID mice of two immunoconjugates, each composed ofa tumor-targeting domain conjugated to the Fc region of a human IgG1heavy-chain, forming a homodimeric molecule similar to a Camelidheavy-chain antibody. For one type of immunoconjugate, thetumor-targeting domain was the human scFv molecule G71-1 that binds tothe melanoma antigen MCSP expressed by the melanoma cells in thexenografts. For the other type of immunoconjugate, the tumor-targetingdomain was a mouse factor VII molecule that binds specifically andtightly to tissue factor (TF), both to mouse TF expressed by the tumorvasculature endothelial cells and to human TF expressed by the melanomacells in the xenografts. To decrease the risk of disseminatedintravascular coagulation (DIC) that might result from the binding of afactor VII immunoconjugate to TF, an active site mutation was introducedinto the mouse fVII targeting domain (mfVIIasm), inhibiting theproteolytic activity required to initiate the blood coagulation pathway.

The study reported in Example 1 showed that the G71-1 immunoconjugatemediates cytolysis of cultured human melanoma cells by NK cells andcomplement. Because SCID mice retain the capacity to produce functionalNK cells and complement, the immunoconjugates could also mediatecytolysis of the targeted tumor cells and vascular endothelial cells ofa human melanoma xenograft growing in SCID mice. Systemic delivery ofthe immunoconjugates to SCID mice was achieved by tail vein injectionsof a replication-defective adenoviral vector encoding theimmunoconjugates, which were secreted into the blood for at least oneweek after each injection. The mice first were injected subcutaneouslywith a human melanoma cell line that expresses either a low or highlevel of TF, and the resulting xenograft was allowed to grow into asmall (˜5 mm³) or larger (˜50 mm³) tumor before starting injections ofthe adenoviral vectors. Further growth of all the xenografts wasprevented for the 3 to 4 week period of the experiments by multipleinjections of the adenovirus encoding the mfVIIasm immunoconjugate,administered separately or together with the adenovirus encoding theG71-1 immunoconjugate; in some of the mice the xenograft completelyregressed. In the control mice, which were injected with an adenovirusthat did not encode an immunoconjugate, the average volume of thexenografts increased by a factor of about 25 during the same period. Inthe mice receiving 5 injections of the adenoviral vectors encoding theimmunoconjugates, many of the liver cells were enlarged and the spleenwas infiltrated with erythrocytes. The defects were not caused by thesecreted immunoconjugates, which do not bind to the liver or spleencells. The primary cause probably is the continuous high level synthesisof the encoded immunoconjugates by the liver cells, which are the mousecells predominately infected by intravenously-injected adenoviralvectors. Although the immunoconjugate concentration in the blood of SCIDmice injected with an adenoviral vector was about 100-fold higher forthe G71-1 immunoconjugate than for the mfVIIasm immunoconjugate, theinhibitory effect on a human melanoma xenograft nevertheless wasstronger with the mfVIIasm immunoconjugate. A key advantage of themfVIIasm immunoconjugate is the binding that occurs to tumor vascularendothelial cells as well as to tumor cells, in contrast to the G71-1immunoconjugate which binds only to melanoma cells. The binding to thetumor vasculature should be tumor-specific, because TF is not expressedby the normal vasculature. Although TF is expressed by several othernormal tissues, such as brain, lung and kidney glomeruli, these TFmolecules are not accessible to endogenous fVII or a fVIIimmunoconjugate because the blood vessel walls form a barrier separatinglarger blood components from adjacent cells. However, tumor bloodvessels are leaky, allowing access to TF expressed by tumor cells. Thus,a human mfVIIasm immunoconjugate can be an effective therapeutic agentfor a broad spectrum of human tumors expressing TF on the vascularendothelial cells and tumor cells. The therapeutic efficacy of a humanmfVIIasm immunoconjugate can be enhanced by also administering a humanscFv immunoconjugate that binds to a tumor cell target other than TF.

Example 3

This example reports a study of the efficacy and safety of a protocolfor cancer treatment tested in a SCID mouse model of human skin andmetastatic lung melanoma, and in an immunocompetent mouse model of mousemelanoma. The protocol involved intratumoral injections ofreplication-incompetent adenoviral vectors encoding immunoconjugates ofthe invention that elicit a cytolytic immune response against thetargeted neovasculature endothelial cells and tumor cells. The mousemodel experiments showed that intratumoral delivery of the factor VIIimmunoconjugate, either alone or together with the single-chain Fvimmunoconjugate, resulted in growth inhibition and regression of theinjected tumor, and also of distant uninjected metastatic tumors,without evidence of damage to normal organs. There was extensivedestruction of the tumor neovasculature, presumably mediated by thefactor VII immunoconjugate bound to tissue factor on neovasculatureendothelial cells. Because tissue factor is generally expressed onneovascular endothelial cells and tumor cells, a factor VIIimmunoconjugate can be used for immunotherapy against a broad range ofhuman tumors.

Materials and Methods. Cell lines. LXSN, TF2 and Yusac2 are humanmelanoma lines, Caki is a human renal tumor line, LnCap is a humanprostate tumor line, A204 is a human neuroblastoma line, B16F10 is amouse melanoma line, EMT6 is a mouse mammary tumor line, BT20 is a humanbreast tumor line, Colo 357 is a human pancreatic tumor line, MS is ahuman gastric tumor line, and 293 is a human kidney line (ATCC,CRL-1573) used for packaging the adenoviral vectors. The culture mediumwas DMEM+10% FBS for all of the tumor lines except LnCap, which wascultured in RPMI 1640+10% FBS.

Adenoviral vectors. 1) Procedures summarized in Example 2 above forproducing and purifying the adenoviral vectors encoding the mfVIIasm andG71-1 immunoconjugates were used with the following modification. Afterthe shuttle vector DNAs were digested with PmeI, instead of an ethanolprecipitation step the DNAs were purified by electrophoresis in agarosegel followed by isolation of the DNA bands using the QIAEX II kit(Qiagen). 2) The vector concentrations in the purified preparations weredetermined by assays for infectious particles (IP) and infectious units(IU), as follows. (i) The IP assay involves diluting the vectorpreparation 20-fold in lysis buffer (0.1% SDS in PBS) and measuringabsorbance at 260 nm; the conversion to IP is 1 O.D. unit=1×10¹² IP.(ii) The IU assay involves infecting cultures of 293 cells with serialdilutions of the vector preparation, incubating the infected culturesfor 2 days, and examining the cells in a fluorescence microscope forexpression of the Green Fluorescent Protein (GFP) gene in the vectorgenome. The IU titer is calculated from the number of cells expressingthe GFP gene. The IP and IU titers agreed within ±10%.

Synthesis of the mfVIIasm immunoconjugate in tumor cells. Tumor cellswere grown almost to confluence in 150-mm dishes, and the cells wereinfected with the adenovirus encoding the mfVIIasm immunoconjugate at amultiplicity of 10 IU per cell. The infected cells were cultured inserum-free medium for 4 days, and 1.5 ml of the medium was mixed with 10μl of a suspension of protein-A beads (Pierce) and rotated at 4° C.overnight. The bound mfVIIasm immunoconjugate was eluted by heating thebeads in 15 μl of SDS-PAGE loading buffer at 80° C. for 3 min, and theeluate was fractionated by SDS-PAGE and transferred to a nitrocellulosemembrane. The immunoconjugate band was detected by immunostaining with agoat anti-human or anti-mouse IgG (Fc specific) probe.

Immunotherapy tests in immunodeficient mice. Female C.B-17 SCID mice 4-to 5-weeks old (Taconic Farms) were used for all experiments withimmunodeficient mice. Monolayer cultures of the human melanoma linesLXSN or TF were dissociated in PBS+2 mM EDTA, washed and resuspended inPBS. Skin tumors were generated by subcutaneous injections of 5×10⁵cells into the right rear flank, and metastatic lung tumors weregenerated by intravenous injections of 6×10⁵ TF cells into the tailvein. The size of the skin tumor was measured in two dimensions with acaliper, and the tumor volume was estimated as (width)2 (length)/2. Whena skin tumor had grown to a volume of about 100 mm3, intratumoralinjections of an adenoviral vector containing a human Fc effector domainwas started. For each injection step, a total volume of 50 μl of thevector preparation was injected into 3 or 4 sites on the tumor. At theend of the experiment, autopsies were done to collect blood samples andto prepare the tumors and normal organs for morphological andhistological examination.

Immunotherapy tests in immunocompetent mice. Female C57BL/6 mice 4- to5-weeks old (Charles River Laboratories) were used for all experimentsinvolving immunocompetent mice. Monolayer cultures of B16F10 mousemelanoma cells were suspended in PBS+2 mM EDTA, washed and resuspendedin PBS. Skin tumors were generated by subcutaneous injections of 5×10⁵cells into the right rear flank. When the skin tumors had grown to anestimated volume of 140 to 325 mm³, tail vein injections of theadenoviral vector encoding the mfVIIasm immunoconjugate containing amouse Fc effector domain were started. The procedures for monitoring theefficacy and safety of the protocol are the same as described above forSCID mice.

SGOT assays. Serum samples were collected from mice, frozen and assayedfor glutamic oxalacetic transaminase using a standard diagnostic kit.

Results. The two immunoconjugates used for this study are composed of atumor-targeting domain and an effector domain (FIG. 1). The targetingdomain is either a mutated mouse factor VII molecule (mfVIIasm) thatbinds to TF expressed on tumor vascular endothelial cells and tumorcells but does not initiate blood coagulation (Example 2), or the scFvantibody G71-1 that binds to its cognate antigen expressed selectivelyon human melanoma cells (Cai and Garen, cited above, and Example 1). Theeffector domain is the Fc region of a human or mouse IgG1 immunoglobulinthat induces a cytolytic immune response against the targeted cells(Example 1). The vector for delivering the immunoconjugates is areplication-incompetent adenovirus encoding a secreted form of theimmunoconjugates (Example 1). In Example 1, intravenous injections intoSCID mice of the vectors encoding the immunoconjugates inhibited tumorgrowth but caused histological damage to the liver, which is the primarytarget for infection by the intravenously injected vector. In order todetermine whether the primary infection could be redirected from livercells to tumor cells, the vectors were injected intratumorally into ahuman skin melanoma growing in SCID mice, and the distribution of thevector in the tumor and liver was mapped by the expression of the GreenFluorescent Protein (GFP) gene inserted into the vector genome (FIG.14). Intense GFP expression was detected in the tumor but not in theliver, indicating that an injected tumor is the primary target forinfection by the vector. The pattern of GFP expression in the tumorappeared to be restricted to a few layers of tumor cells adjacent to thepath traversed by the injection needle.

The effect of vector dose on tumor growth was tested by injecting amixture of the two vectors encoding the mfVIIasm and G71-1immunoconconjugates, into a human melanoma skin tumor growing in SCIDmice, and measuring the tumor volume during the next 5 days. The ratioof the mfVIIasm vector to the G71-1 vector in the mixture was 5 to 1, inorder to compensate for the higher titer of the G71-1 immunoconjugatesecreted into the blood (Example 2). The combined dose of the twovectors used for the injections was varied from 7×10⁸ to 6×10⁹infectious units (IU). For a control, an empty vector without an encodedimmunoconjugate was injected at a dose of 6×10⁹ IU. The strongestinhibition of tumor growth was obtained with the highest dose of thevectors encoding the immunoconjugates. This vector dose was used for allof the following SCID mouse experiments.

In the next experiment, the inhibitory effect on tumor growth ofadministering multiple intratumoral injections of the two vectorsencoding the mfVIIasm and G71-1 immunoconjugates was monitored for 19days. The titer of immunoconjugate proteins in the blood of the mice 2days after the last injection of the adenoviral vectors ranged from 1mg/ml to 2 mg/ml, which is about one-fourth the titer produced byintravenously injected vectors (Example 2). The average volume of thesetumors decreased by about 70% within one day after the first injectionof the two vectors, and the volumes did not subsequently increase duringthe rest of the experiment. Tumors injected with an empty control vectorgrew continuously, reaching at the end of the experiment an averagevolume of 1900 mm³ as compared to 30 mm³ for the tumors injected withthe vectors encoding the two immunoconjugates. Tumors injected only withthe vector encoding the mfVIIasm immunoconjugate were inhibited almostas strongly as the tumors injected with both vectors, consistent withearlier experiments involving intravenous injections of the vectors(Example 2). The mice remained active throughout the experiment, and inautopsies performed 2 days after the final injections all of the organsappeared morphologically and histologically normal and there was noevidence of bleeding. Because SCID mice injected intravenously with thevectors encoding the immunoconjugates showed histological damage to theliver cells (Example 2), the livers of the SCID mice injectedintratumorally were tested for histological and also functional damage.In contrast to the major histological changes seen in liver sectionsfrom the intravenously injected mice, liver sections from theintratumorally injected mice showed relatively minor changes (FIG. 15).Liver function was monitored by assays for the enzyme glutamicoxalacetic transaminase (SGOT) in sera obtained from the mice duringautopsy. The levels of SGOT in the control mice and the mice injectedwith the vectors encoding the immunoconjugates remained within thenormal range (210±28 U/L), indicating that liver function was notimpaired.

The tumors recovered from the mice injected with the vectors encodingthe mfVIIasm and G71-1 immunoconjugates were almost completely devoid ofblood vessels (FIG. 13), presumably as the result of a cytolytic immuneresponse against vascular endothelial cells induced by the mfVIIasmimmunoconjugate bound to TF.

The efficacy of the intratumoral injection protocol depends not only oninhibition of the injected skin tumor but also of metastatic tumors thatare not accessible for injection. To test for an inhibitory effect onmetastatic tumors, we used a SCID mouse model in which human melanoma TFcells are injected into the tail vein, resulting in blood-bornemetastases mainly to the lungs. The mice also were injectedsubcutaneously with human melanoma LXSN cells at the same time, and theskin tumors that formed 12 days later were injected with the vectorsencoding the two immunoconjugates or with an empty control vector. Theintratumoral injections were continued on a biweekly schedule for thenext 8 weeks, and autopsies were performed 2 days after the lastinjection to determine the number of tumor nodules on the surface of thelungs (Table 2). The two mice injected with the control vector had 14and 29 lung nodules, respectively, in contrast to the five mice injectedwith the vectors encoding the two immunoconjugates, three of which hadno lung nodules and two had 1 and 5 nodules, respectively. These resultsindicate that intratumoral injections of the vectors encoding the twoimmunoconjugates can inhibit growth of distant metastatic tumors as wellas the injected tumor.

TABLE 2 Inhibition of metastatic lung tumors by intratumoral injectionsof adenoviral vectors encoding the immunoconjugates. Adenoviral vectorsNumber of metastatic lung tumors Control 14, 29 Encoding theimmunoconjugates 1, 5, 0, 0, 0 SCID mice were injected on day 0 withhuman melanoma cells intravenously to generate blood-borne metastaticlung tumors and subcutaneously to generate a skin tumor. Intratumoralinjections of the adenoviral vectors encoding the mfVIIasm and G71-1immunoconjugates into the skin tumor was started on day 10, andadditional injections were done biweekly for 7 weeks. The autopsies weredone 2 weeks after the last injection. The number of mice was 2 for thecontrol vector and 5 for the vector encoding # the immunoconjugates.

Another parameter that could affect the efficacy of the protocol in aclinical setting is a patient's immune response to the immunoconjugatesor the adenoviral vector. Because the targeting and effector domains ofimmunoconjugates for clinical use would be derived from human proteins,patients should not mount a significant immune response to theimmunoconjugates. However, the adenoviral vector is strongly immunogenicin humans and also in mice. To assess the effect of an immune responseto the adenoviral vector in mice, the protocol was tested inimmunocompetent mice carrying a mouse melanoma skin tumor. Theexperiment involved intravenous instead of intratumoral injections ofthe vector, because the mouse melanoma cells cannot be infected by thevector. Also, only the mfVIIasm immunoconjugate could be tested, becausethe mouse melanoma cells bind the mfVIIasm immunoconjugate but not theG71-1 immunoconjugate. The Fc effector domain of the immunoconjugate forthis experiment was derived from a mouse IgG1 immunoglobulin. Theresults show that intravenous injections of the adenoviral vectorencoding the mfVIIasm immunoconjugate results in growth inhibition of amelanoma tumor in immunocompetent mice as well as in SCID mice (FIG.16).

Because TF is generally expressed by tumor vascular endothelial cellsand also be most metastatic tumor cells, a mfVIIasm immunoconjugatecould mediate a cytolytic immune response not only against humanmelanomas but also against a broad spectrum of other human tumors. Asshown in the study reported here, intratumoral injections of anadenoviral vector encoding the mfVIIasm immunoconjugate into a humantumor appears to be a safe and effective protocol for establishing andmaintaining a high blood titer of the immunoconjugate. However, thecells of the injected tumor must be susceptible to infection by theadenovirus and be capable of synthesizing and secreting the encodedimmunoconjugate. A panel of human and mouse tumor lines, consisting ofhuman melanoma, prostate cancer, breast cancer, pancreatic cancer, renalcancer, gastric cancer, and neuro-blastoma lines and mouse melanoma andbreast cancer lines, was tested as a host for the adenoviral vectorencoding the mfVIIasm immunoconjugate. All of the human tumor linesproduced and secreted about the same amount of the immunoconjugateprotein, indicating that the intratumoral injection protocol could alsobe used for other types of human solid tumors in addition to melanoma.The mouse tumor lines failed to produce the immunoconjugate protein,probably because the mouse cells were not infected by the adenoviralvector.

Discussion. The cancer immunotherapy protocol described in this exampleinvolves intratumoral injections of replication-incompetent adenoviralvectors encoding immunoconjugate molecules that mediate a cytolyticimmune response against the tumor vasculature and tumor cells (FIG. 1).The cells infected by the vectors synthesize the encodedimmunoconjugates, which are secreted into the blood and bind to thecognate targets on tumor vaculature endothelial cells and tumor cells.Intratumoral injection of the vectors provides an important safetyadvantage over intravenous injection, because the vector infectspredominantly the cells of the injected tumor instead of liver cells.Neither the liver nor any other organ of the mice showed significantdamage from repeated intratumoral injections. Several types of humantumor cells other than melanoma also can be infected by the vector andcan synthesize and secrete the encoded immunoconjugate. Thus, theintratumoral injection route for the vectors could be generallyapplicable to human solid tumors. If no tumor is accessible forinjection, the vector could be injected into a susceptible normaltissue.

The therapeutic efficacy of the intratumoral injection protocol wastested in a SCID mouse model of human skin melanoma and metastatic lungmelanoma. The tests involved two immunoconjugates containing differenttargeting domains. 1) One targeting domain is the blood zymogen factorVII (fVII) that binds with high affinity and specificity to thetransmembrane receptor tissue factor expressed by endothelial cells ofgrowing blood vessels, including the vessels of the tumorneovasculature, and also by most human tumor cells. A fVIIimmunoconjugate must compete in vivo with endogenous factor VII forbinding to tissue factor on the targeted cells. This competitionstrongly favors the homodimeric immunoconjugate molecule (FIG. 1) overthe monomeric endogenous molecule, because the avidity effect of twotargeting domains enhances binding to cells expressing multiple copiesof tissue factor (Example 2); also, the blood titer is higher for thevector-encoded fVII immunoconjugate than for endogenous factor VII. Amouse fVII targeting domain was used for the experiments involving humantumor xenografts growing in SCID mice, because it binds tightly both tohuman tissue factor on the tumor cells and to mouse tissue factor on themouse endothelial cells in the tumor vasculature. To prevent initiationof the blood coagulation pathway by the binding of a fVIIimmunoconjugate to tissue factor, which could cause disseminatedintravascular coagulation, a mutation was introduced into the activesite of the mouse fVII targeting domain (mfVIIasm). 2) The secondtargeting domain is the single-chain Fv (scFv) antibody G71-1 that bindsto a chondroitin sulfate proteoglycan expressed selectively by humanmelanoma cells (Examples 1 and 2). The results of the immunotherapytests showed that intratumoral injections of the vector encoding themfVIIasm immunoconjugate, either alone or together with the vectorencoding the G71-1 immunoconjugate, inhibited growth of the injectedskin tumor and also of metastatic lung tumors (FIG. 16 and Table 2). Theresidual tumor tissue remaining after intratumoral injections of thevectors encoding the immunoconjugates was almost devoid of blood vessels(FIG. 13), indicating that the mfVIIasm immunoconjugate induces a potentcytolytic immune response resulting in extensive destruction of thetumor neovasculature.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

The papers, patents, and applications cited herein and the referencescited in them are expressly incorporated in their entireties byreference.

1. An immunoconjugate protein comprising: an Fc domain of a human IgG1immunoglobulin, conjugated to a targeting domain comprising a mutantform of factor VII comprising one or two mutations selected from thegroup consisting of: a substitution of alanine for lysine-341, and asubstitution of alanine for serine-344, wherein the immunoconjugateprotein binds to tissue factor.
 2. An immunoconjugate according to claim1 wherein the targeting domain comprises human factor VII having asubstitution of alanine for lysine-341.
 3. An immunoconjugate accordingto claim 1 wherein the targeting domain of the immunoconjugate proteincomprises human factor VII having a substitution of alanine forserine-344.
 4. An immunoconjugate according to claim 1 wherein theimmunoconjugate protein is made by the process of culturing a cell whichcomprises an expression vector which encodes the immunoconjugate.
 5. Animmunoconjugate according to claim 4 wherein the expression vector is areplication-deficient adenoviral vector.
 6. An immunoconjugate accordingto claim 4 wherein the expression vector is an adeno-associatedexpression vector.
 7. An immunoconjugate according to claim 1 whereinthe immunoconjugate protein forms a dimer of two identical chains, eachhaving said Fc domain of a human IgG1 immunoglobulin and said targetingdomain.
 8. An immunoconjugate according to claim 1 wherein the targetingdomain of the immunoconjugate protein comprises human factor VII havinga substitution of alanine for each of serine-344 and lysine-341.
 9. Theimmunoconjugate protein according to claim 1 which further comprises acytotoxic radioactive tag.
 10. The immunoconjugate protein of claim 1wherein the mutant form of factor VII is native human factor VII havinga substitution of alanine for lysine-341.
 11. The immunoconjugateprotein of claim 1 wherein the mutant form of factor VII is native humanfactor VII having a substitution of alanine for serine-344.
 12. Theimmunoconjugate protein of claim 1 wherein the mutant form of factor VIIis native human factor VII having a substitution of alanine forlysine-341 and for serine-344.